It has been previously shown in our laboratory that antigen-stimulated exocytosis in a mast cell line, RBL-2H3 cell is accompanied by an increased phosphorylation of the heavy (200 kDa) and light (20 kDa) chains of myosin. Both phosphorylations appear to be catalyzed by protein kinase C (PKC). In case of 20 kDa myosin light chain, it has been shown that one site was phosphorylated by myosin light chain kinase (MLCK) in unstimulated cells and de novo site was phosphorylated by PKC upon stimulation by antigen. By using a newly developed one dimensional isoelectrofocusing technique, however, we found that the myosin light chain is phosphorylated at two sites (at serine and threonine residues) by the MLCK. . Stimulation of the cells with antigen increased this phosphorylation in addition to phosphorylation at another serine site by PKC. The phosphorylation by MLCK, which is a calcium-regulated enzyme, may be significant because exocytosis in RBL-2H3 cells is totally dependent on a rise in [Ca2+]i. Therefore, it is plausible that the co-ordination between phosphorylation by MLCK and by PKC is necessary for exocytosis, because some kinase inhibitors (staurosporine, KT5926) equally suppress phosphorylation and secretion, whereas other inhibitors that do not inhibit secretion do not inhibit phosphorylation.